Plant sterols are essential components of all plants. Their functions in plants resemble the functions of cholesterol in mammals. The most abundant plant sterols in the flora are β-sitosterol, campesterol and stigmasterol. The chemical structure of these plant sterols is very similar to that of cholesterol the differences occurring in the side chain of the backbone of the molecule. For example, compared to cholesterol, the side chain of sitosterol contains an additional ethyl group and the side chain of campesterol an additional methyl group.
Since 1950's plant sterols have been known to effectively reduce the serum cholesterol levels. Even when administered in relatively small doses (a few grams a day) they reduce the absorbability of both biliary and dietary cholesterol effectively and thus lower the serum total and LDL-cholesterol levels (12, 28, see also 27, 32). The mechanism by which the restriction of cholesterol absorption happens is still not known in detail, but it is assumed that plant sterols displace cholesterol from the micellar phase and thereby prevent its absorption. In practically all of the early studies, sitosterol or its hydrogenated form sitostanol has been the main plant sterol of interest. However, the sterol composition of the tested preparations has not always been well documented, and the sterol preparations used in most studies have also contained different amounts of other sterols.
Plant sterols have been considered as a safe way of lowering serum cholesterol levels, since they are natural components of vegetable fats and oils. Additionally, their absorption from the intestine of healthy subjects is limited, and the limited amounts absorbed are excreted from the body in the bile. The absorbtion rate of the plant sterols varies between individuals and between the different plant sterols, but for healthy humans usually less than 5% of the plant sterols are absorbed from the digestive tract (27). However, up to 10% of dietary campesterol has been shown to be absorbed (20).
In few rare diseases such as sitosterolemia plant sterols are absorbed exceptionally efficiently, and also the elimination from the body via the biliary route is impaired. Serum levels of sitosterol, campesterol and also their saturated forms sitostanol and campestanol are highly elevated. The elevated levels of the saturated stanols are most probably due to their more effective endogenous synthesis rather than a more effective absorption (10, 27). If untreated, sitosterolemia leads already at young age to xanthomatosis and coronary heart disease. For people with this disease, an administration of unsaturated plant sterols in amounts greater than normally present in foods may lead to hazardous health effects.
Lees and Lees (25) tested the effects of three different sitosterol preparations on plasma lipid and lipoprotein concentrations. One of the preparations was Cytellin, a commercial preparation (Eli Lilly Co., USA) that contained 60-65% sitosterol and 35-40% other sterols, mainly campesterol. An average dose of 18 g/day divided in three doses resulted in a 10.5% average fall in plasma total cholesterol and a 15% fall in LDL-cholesterol. However, when only traces of plant sterols including campesterol are normally detected in plasma (10, 33), the plasma concentration of campesterols varied from 4 to 21 mg/dl in the subjects tested by Lees and Lees (25). In the discussion the authors stated very strongly that since the atherogenicity of campesterol is unknown, the use of a sitosterol preparation with a relatively high campesterol content like the Cytellin preparation used in their study cannot be recommended.
Further, Lees et al. (26) studied the efficacy of plant sterols from soybean oil and tall oil in lowering the blood cholesterol level. They used two different physical forms of each plant sterol, namely a suspension and a powder. The soy sterol consisted of 60-65% sitosterol and 35% campesterol, and a daily dose of an average 18 g of sterols per day (range 9-24 g) was given in three equal doses. A tall oil sterol preparation with only about 5% campesterol was used in this study. A daily dose of 3 grams of both tall oil sterol preparations (powder and suspension) was tested. Additionally, a dose of 6 grams of the tall oil sterol suspension was tested.
Soy sterol in both physical forms and tall oil sterol in powder form reduced the plasma cholesterol content by on average 12% (26). However, the relatively high absorbability of campesterol that has already been shown earlier, was observed also in this study. In the 5 patients tested the plasma campesterol levels ranged from 5 to 21 mg/dl (mean 16 mg/dl). Thus again, even if the cholesterol-lowering effect of soy sterol was proved to be significant, the authors did not recommend its use as a cholesterol-lowering agent. On the contrary, they recommended that pharmaceutical plant sterol preparations should contain a minimum of campesterol and a maximum of sitosterol. Based on the two studies cited above, it can be concluded that the use of vegetable oil based sterols such as soy sterol are strongly not recommendable.
Saturated plant sterols such as sitostanol and campestanol are present in most vegetable oils only in trace amounts. However, tall oil sterols contain 10-15% of sitostanol, the saturated form of sitosterol. Sitostanol can also be made by hydrogenation of the double bond in sitosterol. In the latest studies made with both experimental animals and humans, sitostanol has been proven to be more effective as a cholesterol-lowering agent than sitosterol (8, 16, 17, 18, 19, 36).
An additional advantage of sitostanol is that it is virtually unabsorbable. Several studies (e.g. 9, 16, 17, 21) have shown that sitostanol is practically unabsorbable while small amounts (<5%) of its unsaturated form sitosterol (33) can be absorbed. Similarily, in an in vitro study Amstrong and Carey (6) also showed that cholestanol, a saturated form of cholesterol, was more hydrophobic and less absorbable than cholesterol.
When sitostanol is made by hydrogenation of the most usual plant sterol sources, also another saturated plant sterol, namely campestanol, is formed from campesterol. Until recently, relatively little has been known about the absorbability and the possible hypocholesterolemic effect of this stanol. Based on the data cited above stating that saturated sterols are less absorbable than their unsaturated forms, it could be hypothesized that campestanol might be virtually unabsorbable.
To study the absorbability of different plant sterols Heinemann et al. (20) compared the intestinal absorption of cholesterol with campesterol, sitosterol, stigmasterol and also low concentrations of sitostanol and campestanol in humans by means of intestinal perfusion technique. The results showed that the absorption rate of the differed plant sterols varied between different plant sterols being on average 4.2% for sitosterol, 4.8% for stigmasterol, 9.6% for campesterol and 12.5% for campestanol. Large variation between the absorption efficacy in the ten male subjects was detected.
Thus, according to Heinemann et al. (20) campestanol was found to be more efficiently absorbed than its unsaturated form campesterol. This is against the assumption based on studies cited earlier that showed that the saturated sterols (sitostanol, cholestanol) would be less absorbable than the unsaturated ones (sitosterol, cholesterol). The reason for this remains unclarified. Heinemann et al. (20) speculated, though, that the reason for this conflicting result might be that the study of Amstrong and Carey (6) was made with in vitro conditions and that the theory of the hydrophobicity being a major factor in micellar binding and/or absorption might not be relevant in in vivo conditions. However, this speculation does not explain the fact that several studies that have shown the poorer absorbability of sitostanol compared to that of sitosterol have been made under in vivo conditions. Thus the results of Heinemann et al. (20) that conflict with previous results remained unexplained by the authors.
Sugano at al. (34) studied the hypocholesterolemic activity of corn sterols (composition: 31% campesterol, 4% stigmasterol and 65% sitosterol) and corn stanols (composition: 31% campestanol and 69% sitostanol) obtained by hydrogenation of a corn oil sterol mixture. Two experiments were carried out in rats. Both the sterol and the stenol showed hypocholesterolemic effects at the level of 0.5-1% of the diet when cholesterol (1% in the diet) was ingested. In the first experiment no significant difference was sen in the hypocholesterolemic effect of phytosterols and phytostanols. However, in the second experiment, at the same dietary levels the phytostanols showed considerably greater ability to lower the plasma cholesterol concentration tham did the phytosterols (statistically significant at p<0.02). Moreover, rats fed the 1.0% stanol diet had plasma cholesterol levels significantly lower (p<0.02) than that of the animals fed the diet free of cholesterol. This was not observed in rats fed the 1.0% sterol diet.
Sugano et al. (34) did not study the difference in hypocholesterolemic effect between stanol mixtures with a high content of sitostanol and a low content of campestanol (tall oil sterol based) and stanol mixtures with a substantially higher level of campestanol (vegetable oil sterol based). They compared the hypocholesterolemic effect of an unsaturated sterol mixture with the corresponding saturated stanol mixture. Later studies made by this research group have been focused on the cholesterol lowering effect of sitostanol specificly and compared to sitosterol (21, 22, 23, 35). In fact, in a later publication (23) they refer to the phytostanol study mentioned above (34) mentioning only the hypocholesterolemic effect of β-sitostanol compared to β-sitosterol without discussing any hypocholesterolemic effect of saturated sterols (including campestanol) compared to unsaturated sterols. In the later studies mentioned above sterol mixtures with the typical composition of hydrogenated tall oil sterols with a high content of sitostanol (>90%) have been used.
Miettinen and Vanhanen (30) have shown that sitostanol in fatty acid ester form is more effective than free sitostanol in lowering serum cholesterol levels. Later studies have also shown that the use of sitostanol esters as a part of a daily diet is an effective way of reducing serum total and LDL-cholesterol concentrations (13, 14, 15, 31, 37, 38). The benefit of using stanol esters instead of free stanol is also that the stanol esters are fat-soluble and can therefore easily be incorporated into a wide variety of foods without changing the taste, flavor or physical behavior of the final product. The method for the preparation of sitostanol fatty acid esters and the use of fat-soluble stanol esters in foods have been disclosed in U.S. Pat. No. 5,502,045 (2), hereby incorporated by reference.
Straub (3) suggests the use of saturated stanols (sitostanol, clionastanol, 22,23-dihydrobrassicastanol, campestanol and mixtures thereof) in a method for making a food additive composition where stanols are mixed with an edible solubility agent, an effective amount of a suitable antioxidant and an effective amount of a suitable dispersant. These food additives are intended to reduce cholesterol absorption from foods and beverages which contain cholesterol, e.g. meat, eggs and dairy products. However, in this patent no data showing either any clinical effects or the absorbtion of dietary sterols is presented.
Eugster et al. (1) teach the use of small amounts of sterols, their fatty acid esters and glucosides for the treatment of tumors. The methods of preparation proposed by Eugster et al. involve hazardous chemical reagents like N,N′-carbonyl-diimidazole, thionyl chloride and solvents like tetrahydrofuran, benzen, chloroform or dimethylformamide. Eugster et al. comment on the possible use of these substances as dietary foods and as food additives, but do not present any data on hypocholesterolemic effects or make any claims covering such use. From the disclosure of Eugster et al. it is hard to get a clear picture of how the end product is purified to yield a pure enough sterol ester in large amounts enough to be used as a food component. The only purifying processes referred to are thin layer chromatography and high performance liquid chromathoghaphy. This being the case, the preparation method referred to in the patent by Eugster et al. is limited to small amounts only.
The U.S. Pat. No. 3,751,569 (4) discloses the addition of plant sterol fatty acid esters to cooking oil with the objective of lowering the serum cholesterol levels in man. The patent proposes, for use in the esterification of free sterols, a method which in no case fullfills the requirement for preparation of a food-grade product. According to the patent, the esterification is carried out between a free sterol and a fatty acid anhydride, with perchioric acid acting as a catalyst. The catalyst and reagent used cannot be accepted in food processes. In addition, the patent relates to the fatty acid esters of only native plant sterols. The method proposed in the German patent DE 22 48 921 (5) for the esterification of sterols present in oils and fats by a chemical interesterification technique fullfills the criteria of food processes. In this patent, free sterol and an excess of fatty acid esters are added to a mixture of oil or fat, whereafter the entire fat blend is interesterified by a commonly known interesterification technique. In the resulting fat blend virtually all free sterols have been converted to fatty acid esters. The purpose of this is to protect free sterols in vegetable and animal oils against possible changes during processing.
Earlier data shows that campesterol, one of the major plant sterols, is absorbed relatively efficiently. Therefore it has been recommended that only plant sterol mixtures with a minimum content of campesterol should be used. This has in practice lead to the use of sterol mixtures such as tall oil sterols with a high content of sitosterol.
Most work on stanols has covered sitostanol only. The study of Heinemann et al. (20) showing that campestanol, the saturated form of campesterol, is more readily absorbed than campesterol or sitosterol (12.5%, 9.6% and 4.2% respectively) has lead to a “consensus” that saturated sterol mixtures with “elevated” levels of campestanol are unsafe due to the absorption of campestanol. A clear evidence of this is that all clinical studies covering the use of stanols (sitostanol) have been based on sterol mixtures with a high level of sitostanol and a low level of campestanol.
It is an established fact from many studies (e.g. 8, 17, 18, 19, 23, 36), that sitostanol, the saturated form of sitosterol, is more effective than the corresponding unsaturated sitosterol in reducing the blood cholesterol level. Furthermore saturated sterols are absorbed in very limited amounts, which make the use of saturated sterols a safe mean of reducing cholesterol on a population bases. Of the unsaturated sterols especially campesterol is absorbed in amounts high enough to call for strong recommendations against the use of sterol mixtures with eleveted levels of campesterol (eg. vegetable oil based sterol mixtures) (25, 26).
Accordingly there has been a strong prejudice against using campestanol in any substantial amounts as a substance to be added to foods and this has seriously limited the spectrum of phytosterol containing raw materials to such containing a relatively minor amount of campesterol and its saturated form, campestanol.